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Transcriptome analysis using next generation sequencing reveals molecular signatures of diabetic retinopathy and efficacy of candidate drugs.

Kandpal RP, Rajasimha HK, Brooks MJ, Nellissery J, Wan J, Qian J, Kern TS, Swaroop A - Mol. Vis. (2012)

Bottom Line: These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects.Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA. rkandpal@westernu.edu

ABSTRACT

Purpose: To define gene expression changes associated with diabetic retinopathy in a mouse model using next generation sequencing, and to utilize transcriptome signatures to assess molecular pathways by which pharmacological agents inhibit diabetic retinopathy.

Methods: We applied a high throughput RNA sequencing (RNA-seq) strategy using Illumina GAIIx to characterize the entire retinal transcriptome from nondiabetic and from streptozotocin-treated mice 32 weeks after induction of diabetes. Some of the diabetic mice were treated with inhibitors of receptor for advanced glycation endproducts (RAGE) and p38 mitogen activated protein (MAP) kinase, which have previously been shown to inhibit diabetic retinopathy in rodent models. The transcripts and alternatively spliced variants were determined in all experimental groups.

Results: Next generation sequencing-based RNA-seq profiles provided comprehensive signatures of transcripts that are altered in early stages of diabetic retinopathy. These transcripts encoded proteins involved in distinct yet physiologically relevant disease-associated pathways such as inflammation, microvasculature formation, apoptosis, glucose metabolism, Wnt signaling, xenobiotic metabolism, and photoreceptor biology. Significant upregulation of crystallin transcripts was observed in diabetic animals, and the diabetes-induced upregulation of these transcripts was inhibited in diabetic animals treated with inhibitors of either RAGE or p38 MAP kinase. These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.

Conclusions: Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects. These therapies also changed the abundance of various alternatively spliced versions of signaling transcripts, suggesting a possible role of alternative splicing in disease etiology. Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

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Related in: MedlinePlus

Cyanine (SYBR) green real-time quantitative polymerase chain reaction (qPCR) validation was performed for selected transcripts. The transcripts were amplified as described in Methods section. Three technical replicates were performed for three biologic replicate samples of RNA isolated from nondiabetic animals, diabetic animals and diabetic animals treated with either inhibitor of receptor for advanced glycation endproducts (RAGE) indicated as RI, or an inhibitor of p38 mitogen activated protein kinase (MAPK) designated as p38. The bars (left to right) represent samples corresponding to diabetic animals, diabetic animals treated with RAGE inhibitor, and diabetic animals treated with p38 MAPK inhibitor, respectively. The error bars represent standard deviation from the mean value (n=9). The levels of transcript in diabetic and treated animals were determined relative to the levels present in nondiabetic controls.
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f3: Cyanine (SYBR) green real-time quantitative polymerase chain reaction (qPCR) validation was performed for selected transcripts. The transcripts were amplified as described in Methods section. Three technical replicates were performed for three biologic replicate samples of RNA isolated from nondiabetic animals, diabetic animals and diabetic animals treated with either inhibitor of receptor for advanced glycation endproducts (RAGE) indicated as RI, or an inhibitor of p38 mitogen activated protein kinase (MAPK) designated as p38. The bars (left to right) represent samples corresponding to diabetic animals, diabetic animals treated with RAGE inhibitor, and diabetic animals treated with p38 MAPK inhibitor, respectively. The error bars represent standard deviation from the mean value (n=9). The levels of transcript in diabetic and treated animals were determined relative to the levels present in nondiabetic controls.

Mentions: A few additional transcripts (Cdh3-cadherin, Fgf2-fibroblast growth factor, Lrat-lecithin retinol acyltransferase, Mnd1-meiotic nuclear divisions 1 homolog, Rgr-retinal G-protein coupled receptor, and Sema3c-semaphorin) were chosen for validation with SYBR green based real-time qPCR. All of these transcripts were significantly altered in diabetic mice (Figure 3). As stated for Taqman qPCR validation, the correspondence between RNA-seq and SYBR green qPCR was comparable but not identical.


Transcriptome analysis using next generation sequencing reveals molecular signatures of diabetic retinopathy and efficacy of candidate drugs.

Kandpal RP, Rajasimha HK, Brooks MJ, Nellissery J, Wan J, Qian J, Kern TS, Swaroop A - Mol. Vis. (2012)

Cyanine (SYBR) green real-time quantitative polymerase chain reaction (qPCR) validation was performed for selected transcripts. The transcripts were amplified as described in Methods section. Three technical replicates were performed for three biologic replicate samples of RNA isolated from nondiabetic animals, diabetic animals and diabetic animals treated with either inhibitor of receptor for advanced glycation endproducts (RAGE) indicated as RI, or an inhibitor of p38 mitogen activated protein kinase (MAPK) designated as p38. The bars (left to right) represent samples corresponding to diabetic animals, diabetic animals treated with RAGE inhibitor, and diabetic animals treated with p38 MAPK inhibitor, respectively. The error bars represent standard deviation from the mean value (n=9). The levels of transcript in diabetic and treated animals were determined relative to the levels present in nondiabetic controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351417&req=5

f3: Cyanine (SYBR) green real-time quantitative polymerase chain reaction (qPCR) validation was performed for selected transcripts. The transcripts were amplified as described in Methods section. Three technical replicates were performed for three biologic replicate samples of RNA isolated from nondiabetic animals, diabetic animals and diabetic animals treated with either inhibitor of receptor for advanced glycation endproducts (RAGE) indicated as RI, or an inhibitor of p38 mitogen activated protein kinase (MAPK) designated as p38. The bars (left to right) represent samples corresponding to diabetic animals, diabetic animals treated with RAGE inhibitor, and diabetic animals treated with p38 MAPK inhibitor, respectively. The error bars represent standard deviation from the mean value (n=9). The levels of transcript in diabetic and treated animals were determined relative to the levels present in nondiabetic controls.
Mentions: A few additional transcripts (Cdh3-cadherin, Fgf2-fibroblast growth factor, Lrat-lecithin retinol acyltransferase, Mnd1-meiotic nuclear divisions 1 homolog, Rgr-retinal G-protein coupled receptor, and Sema3c-semaphorin) were chosen for validation with SYBR green based real-time qPCR. All of these transcripts were significantly altered in diabetic mice (Figure 3). As stated for Taqman qPCR validation, the correspondence between RNA-seq and SYBR green qPCR was comparable but not identical.

Bottom Line: These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects.Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA. rkandpal@westernu.edu

ABSTRACT

Purpose: To define gene expression changes associated with diabetic retinopathy in a mouse model using next generation sequencing, and to utilize transcriptome signatures to assess molecular pathways by which pharmacological agents inhibit diabetic retinopathy.

Methods: We applied a high throughput RNA sequencing (RNA-seq) strategy using Illumina GAIIx to characterize the entire retinal transcriptome from nondiabetic and from streptozotocin-treated mice 32 weeks after induction of diabetes. Some of the diabetic mice were treated with inhibitors of receptor for advanced glycation endproducts (RAGE) and p38 mitogen activated protein (MAP) kinase, which have previously been shown to inhibit diabetic retinopathy in rodent models. The transcripts and alternatively spliced variants were determined in all experimental groups.

Results: Next generation sequencing-based RNA-seq profiles provided comprehensive signatures of transcripts that are altered in early stages of diabetic retinopathy. These transcripts encoded proteins involved in distinct yet physiologically relevant disease-associated pathways such as inflammation, microvasculature formation, apoptosis, glucose metabolism, Wnt signaling, xenobiotic metabolism, and photoreceptor biology. Significant upregulation of crystallin transcripts was observed in diabetic animals, and the diabetes-induced upregulation of these transcripts was inhibited in diabetic animals treated with inhibitors of either RAGE or p38 MAP kinase. These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.

Conclusions: Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects. These therapies also changed the abundance of various alternatively spliced versions of signaling transcripts, suggesting a possible role of alternative splicing in disease etiology. Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

Show MeSH
Related in: MedlinePlus